Uncovering low-level mosaicism in human embryonic stem cells using high throughput single cell shallow sequencing

Area of Science:

• Stem Cell Biology
• Genetics
• Genomics

Background:

• Human pluripotent stem cells (hPSCs) exhibit genetic mosaicism, including copy number variations (CNVs).
• Current bulk DNA analysis methods often fail to detect low-grade mosaicism in hPSCs.
• CNVs in hPSCs pose risks for clinical translation due to unknown or dangerous in vivo effects.

Purpose of the Study:

• To develop and validate a high-throughput, cost-effective method for analyzing genetic content in single hPSCs.
• To investigate the frequency and spectrum of genetic aberrations in hPSCs at the single-cell level.
• To assess the feasibility of using single-cell whole genome amplification and shallow sequencing for hPSC genetic analysis.

Main Methods:

• Utilized the Fluidigm C1 single-cell whole genome amplification (WGA) platform.
• Combined WGA with shallow whole genome sequencing (sWGS) for genetic analysis of individual hPSCs.
• Analyzed 56 single hPSCs from a line carrying an isochromosome 20.

Main Results:

• Identified 50 genetic aberrations across 23% of analyzed single hPSCs, undetectable by bulk analysis.
• Aberrations primarily consisted of segmental gains, with fewer segmental losses and aneuploidies.
• Observed that 40% of identified breakpoints correspond to known DNA fragile sites.

Conclusions:

• Demonstrated the feasibility of single-cell shallow sequencing for analyzing hPSC genetic content.
• Confirmed the significant prevalence and biological importance of single-cell genetic mosaicism in hPSCs.
• Highlighted the necessity of single-cell analysis for accurate detection of hPSC genetic aberrations for safe clinical application.